CN114887063B

CN114887063B - Application of Pacsin1 in inhibition of remifentanil-induced hyperalgesia

Info

Publication number: CN114887063B
Application number: CN202210461447.3A
Authority: CN
Inventors: 李依泽; 张麟临; 李楠; 康佳敏; 王国林; 于泳浩
Original assignee: Tianjin Medical University General Hospital
Current assignee: Tianjin Medical University General Hospital
Priority date: 2022-04-28
Filing date: 2022-04-28
Publication date: 2023-05-23
Anticipated expiration: 2042-04-28
Also published as: CN114887063A

Abstract

The invention discloses an application of Pacsin1 in inhibiting remifentanil-induced hyperalgesia. Experiments prove that after Pacsin1 gene knockout, the hyperalgesia induced by remifentanil can be greatly relieved. The results of the present invention thus provide a new therapeutic regimen for remifentanil-induced hyperalgesia in the clinic.

Description

Application of Pacsin1 in inhibition of remifentanil-induced hyperalgesia

Technical Field

The invention belongs to the field of biological medicine, and relates to an application of Pacsin1 in inhibiting hyperalgesia induced by remifentanil.

Background

Opioids are the most important analgesic drugs for clinical treatment of acute and chronic pain and cancer pain, and the clinical dosage is extremely large, but the opioids can also activate the nociceptive mechanism in the body while relieving pain, which is manifested by the enhancement of the body's responsiveness to nociceptive stimuli and the increase of the demand of analgesic drugs, namely opioid-induced hyperalgesia (opioid-induced hyperalgesia, OIH). Remifentanil is an ultra-short mu-opioid receptor agonist, and is widely applied to analgesia in clinical operations due to the advantages of quick effect, quick clearance, no accumulation, no metabolic dependence on liver and kidney functions and the like. However, remifentanil induces postoperative hyperalgesia (RIH) at a much higher rate than other opioid analgesics, up to 85%. Another study found that RIH occurred at 32.7% in patients with surgery time exceeding 2 hours, and at cumulative infusion exceeding 30 μg/kg, RIH occurred at even up to 41.8%. RIH is mainly characterized by an increase in the extent and range of postoperative incision pain that occurs after infusion of remifentanil at a rate of 0.05-0.3 μg/kg/min for 60-90 min, and an increase in opioid analgesic demand. RIH not only reduces the analgesic effect of the medicine, but also promotes pain perception, produces abnormal pain, even causes postoperative chronic pain, patients have larger and larger dosage requirements for opioid medicines, not only increases hospitalization time, medical cost and occupies medical resources, but also most importantly increases physical and psychological trauma of the patients, aggravates the pain of the patients and seriously influences the life quality of the patients. At present, no effective treatment measures exist clinically, mainly because the occurrence mechanism is not clear at present, so that it is urgent to deeply elucidate the pathogenesis of remifentanil-induced hyperalgesia and to find effective treatment strategies.

Disclosure of Invention

The invention provides application of an agent for inhibiting Pacsin1 in preparation of a medicament for preventing or treating opioid-induced postoperative hyperalgesia.

Further, the opioid is remifentanil.

Further, the agent that inhibits Pacsin1 includes an agent that inhibits Pacsin1 expression.

Further, the agent that inhibits expression of Pacsin1 includes an agent that inhibits expression of Pacsin1 gene mRNA and/or an agent that inhibits expression of Pacsin1 protein.

Further, the agent for inhibiting Pacsin1 expression includes an agent for inhibiting Pacsin1 gene expression by gene knockout technique, antisense nucleotide technique, RNAi technique.

Further, the gene knockout technology comprises CRISPR technology, zinc finger enzyme ZFN technology and TALEN technology.

Preferably, the gene knockout technique uses CRISPR technique;

more preferably, CRISPR technology uses a CRISPR/Cas system;

in a specific embodiment of the invention, the CRISPR/Cas system is a CRISPR/Cas9 system.

Methods for introducing a nucleic acid molecule or expression vector thereof into a cell useful in the present invention include, but are not limited to: viral transduction, microinjection, particle bombardment, gene gun transformation, electrotransfection, liposome transfection, and the like.

Further, the RNAi technology inhibits Pacsin1 gene expression using reagents including the following: double stranded RNA, short hairpin RNA or microRNA.

Further, hyperalgesia includes mechanical hyperalgesia and thermal hyperalgesia.

Further, the dosage form of the drug is any pharmaceutically acceptable dosage form.

The dosage forms of the medicine include, but are not limited to, tablets (including dispersible tablets, enteric-coated tablets, chewable tablets, orally disintegrating tablets, effervescent tablets and the like), hard capsules (including enteric-coated capsules), soft capsules, granules, pills, micropills, dripping pills, dry suspensions, oral solutions, dry syrups, powders, oral suspensions, oral quick-release or slow-release or controlled-release dosage forms and the like, injections (including injectable powder injections (including injectable sterile filling powders, freeze-dried powder injections), aqueous solution injections), ointments, gels, emulsions, emulsion, patches, suppositories, gels and the like.

The medicaments of the invention can be applied by oral route or by intravenous, intramuscular, intradermal or subcutaneous injection.

The medicine of the invention can be used alone or in combination with other medicines for treating opioid-induced postoperative hyperalgesia.

The medicaments of the present invention also include pharmaceutically acceptable ingredients including, but not limited to, pharmaceutically acceptable carriers, diluents, excipients, adjuvants, buffers, pH adjusting agents, preservatives, antioxidants, bacteriostats, stabilizers, suspending agents, solubilizing agents, surfactants (e.g., wetting agents), colorants, and isotonicity solutes (i.e., that render the formulation isotonic with the blood or other related bodily fluids of the subject patient). Suitable carriers, diluents, excipients, and the like can be found in standard pharmaceutical books. See, e.g., handbook of pharmaceutical additives (Handbookof Pharmaceutical Additives), second edition (editors m.ash and i.ash), 2001 (SynapseInformation Resources, inc., endicott, newYork, USA); remington' sPharmaceutical Science, 18 th edition, mack Publishing Company, easton, pa.,1990; and handbooks of pharmaceutical excipients (Handbook ofPharmaceutical Excipients), second edition, 1994.

The term "pharmaceutically acceptable" as used herein relates to compounds, ingredients, materials, compositions, dosage forms, and the like, which are, within the reasonable scope of medical judgment, suitable for contact with the tissues of patients without undue unwanted toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.

The invention also provides a pharmaceutical composition for preventing or treating opioid-induced postoperative hyperalgesia, comprising an agent that inhibits Pacsin 1.

Preferably, the agent that inhibits Pacsin1 comprises an agent that inhibits Pacsin1 expression;

preferably, the agent that inhibits expression of Pacsin1 comprises an agent that inhibits expression of Pacsin1 gene mRNA and/or an agent that inhibits expression of Pacsin1 protein;

preferably, the agent that inhibits expression of Pacsin1 comprises an agent that is used in the process of inhibiting expression of Pacsin1 gene using gene knockout technology, antisense nucleotide technology, RNAi technology.

The invention also provides a method of preventing or treating opioid-induced postoperative hyperalgesia comprising administering to a subject an effective amount of an agent that inhibits Pacsin1 as described previously.

The term "effective amount" as used herein refers to an amount of a compound or composition sufficient to treat a specified disorder, condition, or disease, such as to ameliorate, alleviate, mitigate, and/or delay one or more symptoms thereof.

The "subject" of the invention may be an animal, mammal, placental mammal, rodent (e.g., guinea pig, hamster, rat, mouse), murine (e.g., mouse), lagomorph (e.g., rabbit), canine (e.g., dog), feline (e.g., cat), equine (e.g., horse), porcine (e.g., pig), ovine (e.g., sheep), bovine (e.g., cow), primate, simian (e.g., monkey or ape), monkey (e.g., marmoset, baboon), ape (e.g., gorilla, chimpanzee, gorilla, gibbon), or human.

The term "vector" as used herein includes any intermediate vector for a nucleic acid molecule that enables the nucleic acid molecule to be introduced into a prokaryotic and/or eukaryotic cell and/or integrated into the genome, and includes plasmids, phages, phage or viral vectors, such as retroviral vectors, adeno-associated viral vectors, and the like.

The terms "CRISPR/Cas", "CRISPR system" or "CRISPR-Cas system" as used herein are collectively referred to as transcripts and other elements involved in expressing or directing CRISPR-associated gene (Cas) activity, including nucleic acids encoding Cas genes, tracr (transactivation CRISPR) sequences (e.g., active portion tracrRNA), tracr-mate sequences (comprising "direct repeat" and direct repeat of the portion of the tracrRNA process in the context of endogenous CRISPR systems), guide sequences (grnas, e.g., RNAs for guiding Cas, e.g., cas9; CRISPR RNA and transactivation (tracer) RNAs or single guide RNAs (sgrnas)), or other sequences and transcripts from the CRISPR locus. CRISPR-Cas is optionally a class II monomeric Cas protein, such as a type II Cas or a type V Cas. The Cas protein type II may be a Cas9 protein, for example Cas9 from streptococcus pyogenes (Streptococcus pyogenes), franciscensis neoformans (Francisella novicida), actinomyces naesulndii (a.naesulndii), staphylococcus aureus (Staphylococcus aureus) or Neisseria meningitidis (optiona). Preferably, cas9 is from streptococcus pyogenes. The V-type Cas protein may have RNA processing activity. The type V Cas protein may be a Cas12a (also known as Cpf 1) Cas protein, such as Cas12a (Lb-Cas 12 a) from Lachnospiraceae bacteria or from acidococcus sp.bv3l6 (as-Cas 12 a). The CRISPR system may also be a CRISPR-Cpf1 system, wherein Cas such as Cas9 is replaced by Cpf 1. A typical feature of CRISPR systems is the elements that promote the formation of CRISPR complexes at target sequence sites.

The term "gRNA" or "guide RNA" as used herein refers to an RNA molecule that hybridizes to a particular DNA sequence (e.g., crRNA), and also includes protein binding fragments that bind to a CRISPR-Cas protein called tracrRNA. The gRNA may also include direct repeat sequences. The portion of the guide RNA that hybridizes to a particular DNA sequence is referred to herein as a nucleic acid targeting sequence, or crRNA or spacer sequence. As will be understood from the context, gRNA may also refer to or be represented by a corresponding DNA sequence encoding gRNA. Since the target specific portion or crRNA can bind to different tracrRNA, the guide sequences provided herein minimally include crRNA sequences.

The term "crRNA" also referred to as "spacer sequence" or including spacer sequences as used herein refers to a portion of a gRNA that forms or is capable of forming an RNA-DNA duplex with a target sequence. The sequences may be complementary or correspond to a particular CRISPR target sequence. The nucleotide sequence of the CrRNA/spacer sequence can determine the CRISPR target sequence and can be designed to target a desired CRISPR target site. CrRNA can also refer to or be represented by the corresponding DNA sequence encoding CrRNA as understood from the context.

Drawings

Fig. 1 shows a graph of the results of Pacsin1 inhibiting pain sensitivity of incisions after remifentanil infusion, wherein a: different sets of mechanical stimulus-induced foot contraction frequencies (%); b: foot withdrawal latency (sec) for different groups of thermal stimuli; NS: continuously infusing normal saline with the same volume as remifentanil through the abdominal cavity for 60min; RI: continuous infusion of remifentanil 1 μg kg via abdominal cavity ^-1 ·min ^-1 60min, and simultaneously establishing an incision pain model; c+ri: cas9KO plasma id 1 μl was injected 1 month before remifentanil was injected into L4-5 dorsal root ganglion, and remifentanil 1 μg kg was continuously infused intraperitoneally ^-1 ·min ^-1 60min, and simultaneously establishing an incision pain model; pko+ri: pacsin1-Cas9KO plasma+remifentanil+incision pain group, pacsin1-Cas9KO plasma 1 μl was injected into L4-5 dorsal root ganglion 1 month before remifentanil was injected, and remifentanil 1 μg kg was continuously infused through abdominal cavity ^-1 ·min ^-1 60min, and simultaneously establishing an incision pain model;

FIG. 2 shows a graph of ELISA results;

note that: n=10; * P <0.001 compared to NS group; p <0.001, compared to RI group; two-way ANOVA.

Detailed Description

The invention will now be described with reference to the following examples, which are intended to illustrate the invention, but not to limit it.

The experiments and methods described in the examples were performed substantially in accordance with conventional methods well known in the art and described in various references unless specifically indicated. The specific conditions are not noted in the examples and are carried out according to conventional conditions or conditions recommended by the manufacturer. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention. Those skilled in the art will appreciate that the examples describe the invention by way of example and are not intended to limit the scope of the invention as claimed. All publications and other references mentioned herein are incorporated by reference in their entirety.

Example Pacsin1 and remifentanil-induced hyperalgesia correlation study

1. Experimental method

(1) Experimental grouping: male SD mice were purchased from the experimental animal center of the military medical science sciences of the civil liberation army of china 24, 1 month old. The random number table method was used to divide into 4 groups (n=6):

1) Saline group (NS group), continuously infusing the same volume of saline as remifentanil through the abdominal cavity for 60min;

2) Remifentanil + incision pain group (RI group) was continuously infused with remifentanil 1 μg kg through the abdominal cavity ^-1 ·min ^-1 60min, and simultaneously establishing an incision pain model;

3) Cas9KO plasma + remifentanil + incision pain group (C + RI group): cas9KO plasma id is purchased from Santa Cruz Biotechnology under the trade name sc-418922, and Cas9KO plasma id 1 μl is injected into L4-5 dorsal root ganglion 1 month before remifentanil is injected, and remifentanil 1 μg.kg is continuously infused through abdominal cavity ^-1 ·min ^-1 60min, and simultaneously establishing an incision pain model;

4) Pacsin1-Cas9KO plasma+remifentanil+incision pain group (PKO+RI group): pacsin1-Cas9KO plasmid is purchased from Santa Cruz Biotechnology under the trade name SC-423925 and injected into 1 μl of Pacsin1-Cas9KO plasmid at L4-5 dorsal root ganglion 1 month before remifentanil is injected, and remifentanil 1 μg.kg is continuously infused through abdominal cavity ^-1 ·min ^-1 Totally 60min, simultaneously establishing an incision pain model.

(2) Incision pain model preparation: a model of incision pain was prepared. The mice were anesthetized with 2% sevoflurane, left hind feet were sterilized, longitudinal incisions were made about 0.5cm long from the proximal end of the sole to the toe, after skin was incised, plantar muscles were lifted with an ophthalmic forceps and the longitudinal separation to periosteum was maintained, and the starting and stopping of the muscles and attachment were intact. After hemostasis by compression, the skin was sutured with 4-0 silk threads. The incision skin cannot overlap, invert, split. The wound is disinfected by iodophor and a small amount of erythromycin ointment is smeared to prevent infection.

(3) Behavioural experiments: thermal stimulation Paw Withdrawal Latency (PWL) and mechanical stimulation Paw Withdrawal Frequency (PWF) were measured 24h before infusion of remifentanil (T0), 2, 6, 24 and 48h after cessation of infusion (T1-4), laboratory temperatures were 18-22 ℃, and were quiet. The PWL was measured by YLS-6B intelligent hotplate instrument (Huai North Zhenghua biological instrument Co., ltd.), the time from the left hindfoot contact with the hotplate to the occurrence of any one of the reactions of retraction, standing on the foot, struggling, hoarseness and licking was recorded as PWL, and the measurement was continued 3 times at 5min intervals, and the average value was taken as PWL (sec). To prevent scalding the paw, the PWL upper limit was set to 20s. The rats were placed in a 20cm x 20cm metal cage, after 30min, were stimulated with 0.4g (Harvard Apparatus company, U.S.) of BSEVF3 von Frey fiber yarn between the 2, 3 phalanges of the right hind paw, pressure was applied vertically, the pressure was recorded as the onset of rapid paw withdrawal response, licking the right paw or fizzing, and the pressure was measured continuously 10 times, 1min apart, with the paw withdrawal frequency being PWF (%).

(4) ELISA: after the end of the last 1 behavioural assay, mice were sacrificed and L4-5 dorsal root ganglion was taken and the expression of Pacsin1 was determined by ELISA. Adding precooled tissue protein lysate into dorsal horn tissue of spinal cord, and grinding into tissue homogenate. Centrifuging the homogenate at 4 ℃ for 5min at 12000rpm with a radius of 10cm, and obtaining the supernatant as the total protein of the spinal cord tissue. The membrane protein was extracted by specific procedures according to the instructions using a membrane protein extraction kit (Thermo company, usa). The expression of Pacsin1 protein was determined experimentally using Pacsin1 ELISA Kit (Santa Cruz Biotechnology, SC-423925, USA) following the guidelines of the specification.

(5) Statistical analysis: the SPSS 18.0 statistical software is adopted for analysis, the normal distribution measurement data is expressed by mean ± standard deviation (±s), the measurement data of the random block design is compared by single factor analysis of variance, the measurement data of the repeated measurement design is compared by repeated measurement design analysis of variance, and P <0.05 is the difference and has statistical significance.

2. Experimental results

(1) Remifentanil infusion exacerbates post-operative mechanical and thermal hyperalgesia

Remifentanil + incision pain (RI) group at 1 μg kg compared to saline (NS) group infusion ^-1 ·min ^-1 Is infused for 60 minutes at a rate of from 2h to 48h, resulting in a significant increase in the foothold frequency (PWF) and a significant decrease in the foothold latency (PWL) (all P's)<0.001). These results indicate that the concentration is 1. Mu.g.kg ^-1 ·min ^-1 Is effective in increasing the thermal and mechanical hyperalgesia of the incision caused by opioids. Hypersensitivity to incision-induced thermal and mechanical pain caused by remifentanil infusion and incision pain model (RI group) can last from 2 hours to 48 hours (fig. 1A and 1B).

(2) Pacsin1 can inhibit incision pain sensitivity after remifentanil infusion

Pacsin1 knockdown significantly reduced mechanical hyperalgesia (FIG. 1A, the paw withdrawal frequency characterizes mechanical pain) and thermal hyperalgesia (FIG. 1B, the paw withdrawal latency characterizes thermal pain) induced by remifentanil incision pain, suggesting a potential analgesic property for Pacsin1 knockdown.

(3) Remifentanil infusion and incision increased dorsal root ganglion Pacsin1 expression

Mice were sacrificed to remove dorsal root ganglion 48h after remifentanil and incision pain models, and significant increases in Pacsin1 protein expression were found in ELISA results (P <0.001, fig. 2). The above results indicate that hyperalgesia following remifentanil infusion is associated with elevated expression of Pacsin1 in dorsal root ganglion.

Although specific embodiments of the invention have been described in detail, those skilled in the art will appreciate that: many modifications and variations of details may be made to adapt to a particular situation and the invention is intended to be within the scope of the invention. The full scope of the invention is given by the appended claims together with any equivalents thereof.

Claims

1. Use of an agent that inhibits Pacsin1 in the manufacture of a medicament for preventing or treating remifentanil-induced post-operative hyperalgesia; the reagent for inhibiting Pacsin1 is a reagent for inhibiting Pacsin1 expression by a gene knockout technology, and the gene knockout technology is a CRISPR Cas9 technology.

2. The use according to claim 1, wherein hyperalgesia comprises mechanical hyperalgesia and thermal hyperalgesia.

3. The use according to claim 1, wherein the dosage form of the medicament is any pharmaceutically acceptable dosage form.